Umbilical cord mesenchymal stromal cells transplantation delays the onset of hyperglycemia in the RIP-B7.1 mouse model of experimental autoimmune diabetes through multiple immunosuppressive and anti-inflammatory responses

Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet beta cells. Despite many efforts focused on identifying new therapies able to counteract this autoimmune attack and/or stimulate beta cells regeneration, TD1M remains without effective clinical treatments providing no clear advantages over the conventional treatment with insulin. We previously postulated that both the inflammatory and immune responses and beta cell survival/regeneration must be simultaneously targeted to blunt the progression of disease. Umbilical cord-derived mesenchymal stromal cells (UC-MSC) exhibit anti-inflammatory, trophic, immunomodulatory and regenerative properties and have shown some beneficial yet controversial effects in clinical trials for T1DM. In order to clarify conflicting results, we herein dissected the cellular and molecular events derived from UC-MSC intraperitoneal administration (i.p.) in the RIP-B7.1 mouse model of experimental autoimmune diabetes. Intraperitoneal (i.p.) transplantation of heterologous mouse UC-MSC delayed the onset of diabetes in RIP-B7.1 mice. Importantly, UC-MSC i. p. transplantation led to a strong peritoneal recruitment of myeloid-derived suppressor cells (MDSC) followed by multiple T-, B- and myeloid cells immunosuppressive responses in peritoneal fluid cells, spleen, pancreatic lymph nodes and the pancreas, which displayed significantly reduced insulitis and pancreatic infiltration of T and B Cells and pro-inflammatory macrophages. Altogether, these results suggest that UC-MSC i. p. transplantation can block or delay the development of hyperglycemia through suppression of inflammation and the immune attack.

Splenocytes were gated on the basis of their SSC-H and FSC-H characteristics (blue gate in SSC-H/FSC-H dot plots). Right dot plots in panels show CD11b and Ly6C coexpression levels of R1 gated splenocytes. CD11b + were divided in 3 subpopulations based on their differential expression levels of Ly6C. (b) Shows SSC-H and FSC-H subpopulations analysis in splenocytes from IMM-RIP-B7.1 mice at 7 days after UC-MSC transplantation. Subpopulations could be identified as corresponding to CD11b + /Ly6Cmacrophages (red dots), CD11b + /Ly6C med granulocytic MDSC (blue dots) and CD11b + /Ly6C high monocytic MDSC (dark dots), distinguishable in size and complexity from splenic lymphocytes (grey dots).

Peritoneal drainages
For peritoneal drainages collections, mice were killed by cervical dislocation and abdominal skin was removed with the use of scissors. The exposed abdominal wall was then gently sterilized with ethanol and dried. Mice were then fixed side down on the border of a platform. An untreated P80 sterile Petri dish was placed on ice at a distance of 10 centimeters below the mouse. A total volume of 8-10 ml cold PBS solution containing 2% BSA and 2.5 mM EDTA was then injected into the peritoneal cavity with the use of a 21G needle. Special care was taken to not damage visceral organs during injection of the PBS solution. A gentle massage of the abdomen was done just before performing a small incision in the center of abdominal wall. PBS solution containing peritoneal immune cells was then gently collected in the Petri dish. The use of EDTA and non-adherent petri dish to collect peritoneal lavage cells was found to be of critical importance to avoid adherence of peritoneal macrophages. PBS solution containing peritoneal lavage cells was then directly transferred to 15 ml polystyrene conical tube on ice.

Splenocytes isolation
Spleen were surgically removed from the sacrificed mice just after peritoneal drainages and were transferred into cold PBS+2%BSA+2.5mMEDTA solution on ice. Spleens were then placed between the frosted ends of two glass slides and mechanically disrupted with constant resuspension of the cells in the cold PBS solution. The solution of splenocytes was then filtered through a 100 µm cell strainer (BD Falcon) placed into a 50ml conical tube on ice and the resulting cell suspension was then centrifuged at 4ºC and cells subjected to erythrocytes lysis.
The resulting splenocytes pellet was finally resuspended into 1 ml of cold PBS+2%BSA+2.5mMEDTA solution and aliquots used for labelings with conjugated antibodies for flow cytometry analysis or for qPCR analysis.

Pancreatic lymph nodes isolation
Pancreases were then surgically removed together with the mesentery and placed into a Petri dish containing cold PBS+2%BSA+2.5mMEDTA solution on ice. The connected pancreas and mesentery were then surgically separated under magnification and with the use of a small scissor and precision tweezers, pancreatic lymph nodes were removed and transferred into a clean Petri dish placed on ice and containing cold PBS+2%BSA+2.5mMEDTA solution. The isolation of pancreatic lymph nodes cells was then performed almost identically as described previously for splenocytes isolation, with the use of the frosted ends of glass slides. Pancreatic lymph nodes cells were not subjected to erythrocytes lysis and directly centrifuged at 4ºC, prior being used for antibodies labelings or RNA extraction for qPCR analysis.

Pancreatic stromal cells isolation
Following the collection of pancreatic lymph nodes, pancreases were washed in cold PBS checked again under magnification for ensuring total removal of pancreatic lymph nodes. Pancreases were then transferred to 15 ml conical polystyrene tube containing 3 ml pre-warmed DMEM low glucose basal containing 2 mg/ml collagenase type I (Sigma-Aldrich C0130) and 1 mg/ml collagenase type V (Sigma-Aldrich C9263) + 1%BSA. Digestion samples were gently shaken 2 to 3 times during the digestion procedure that usually last 15 to 20 minutes. Digested pancreases were then filtered onto 100 µm cell strainer (BD Falcon) to collect the resulting cell suspensions that was finally centrifuged and resuspended into cold PBS+2%BSA+2.5mMEDTA solution and placed on ice until use for antibodies labelling or extraction of RNA.